Proteins are biopolymers which are dependent upon structural stability to enact their specified function. Since a small change in solvent composition, pH, temperature, and salt concentration can often exert a significant and occasionally irreversible change in protein conformation, chromatographic protein purification has ideally been performed using resins exhibiting minimal non-specific, denaturing interactions. Classically, such resins have been extremely hydrophilic, having a water content often exceeding 80%. As a result of their hydrophilic nature, the resultant chromatographic resin particles are most susceptible to collapse, even under modest back-pressure. In addition, any non-specific adsorption can be difficult to displace due to an inability to effectively wash these hydrophilic resins with organic solvents. Consequently, one is confronted with a problem in the initial step of preparative purification of proteins from heterogeneous natural sources. The more desirable supports, due to their hydrophilic nature, are inappropriate for rapid throughput of viscous, sludge-laden, natural product mixtures. As a result, it has been necessary to use, at considerable additional expense, non-chromatographic methods for initial purification.
Amberlite.RTM. XAD resins are polymeric macroreticular adsorbents, commercially produced by the Rohm and Haas Company. These resins have been designed for the separation of compounds based upon the varied affinity of the latter for a polymeric hydrophobic surface. Since XAD-type resins (1) have a large particle size (20-50 mesh) and (2) are extremely hydrophobic, any practical utilization of such resins in the chromatography of complex mixtures of structurally similar peptides and proteins would be surprising. Indeed, there is no report which details the operational parameters of these supports in protein purification. However, it is the foregoing two properties of the XAD-type resins which surprisingly make them exceptionally effective for the initial purification stages of highly impure sludge-laden mixtures containing both structurally diverse and structurally similar proteins. One would correctly expect that the large and heterogeneous particle sizes of XAD-type resins would substantially diminish their chromatographic performance due to the slow and unequal dynamics of interaction and, therefore, one would avoid the use of such resins in protein and polypeptide purification. It has been discovered, however, that this seeming deficiency in fact serves as an advantage when applied under precisely defined conditions to highly impure, sludge-laden materials containing proinsulin-like material.
Moreover, of added practical significance in the purification of such proinsulin-like material is the fact that XAD-type resins (1) are readily available at moderate cost, (2) are completely stable throughout the pH range of 1-13, and (3) are amenable to in-column regeneration with aqueous detergents and organic solvents.
The literature does not address, except in a most general manner, the use of XAD-type resins in the purification of proteins and polypeptides. Thus, for example, technical bulletins provided by the Rohm and Haas Company discuss adsorption of proteins on XAD-7 resin but fail to provide any enlightenment regarding the conditions of separation or efficiency of operation. Pietrzyk, D. J. and Stodola, J. D., Anal. Chem. 53, 1822-1828 (1981) were the first to analytically examine XAD-4, a co-polymer of polystyrene-divinylbenzene, for utilization with synthetic dipeptides. A further study [Pietrzyk, D. J., Cahill, W. J., and Stodola, J. D., J. Liquid Chrom. 5, 443-461 (1982)]with synthetic peptides as large as five residues revealed the possibility of achieving reasonably efficient preparative purification on XAD-4 resin which first had been crushed and sized to significantly smaller particles. Consequently, while these studies did indicate the ability to effectively chromatograph small peptides on macroporous hydrophobic resins, they did not address the question whether mixtures of substantially larger and vastly more complex proteins could be efficiently separated from highly impure mixtures using large particle size supports.
The difficulties of protein purification from highly impure sources have been especially evident with the advent of recombinant DNA technology and its particular suitability to the commercial production of peptides and proteins. Any commercially feasible expression of product by recombinant DNA methodology necessarily carries with it the requirement to isolate the recombinant DNA-sourced product from impurities contained in the originating fermentation broths as well as in the mixtures resulting from subsequent chemical and/or other treatments. The necessity for new commercial-scale protein purification methodology thus has become a high priority item.
An even more complicating factor in the purification of recombinant DNA-sourced proteins arises from the presence in many such proteins of cysteinyl residues. In most cases, following recombinant expression of cysteine-containing proteins, the cysteinyl sulfhydryls must be reversibly protected, generally by conversion to S-sulfonates, prior to commencing any protein purification. This essential conversion necessarily leads to the production of additional amounts of undesirable sludge-like impurities, in the presence of highly viscous denaturing agents, from which the desired protein must first be separated.
As a specific example, recombinant DNA-source insulin is available generally via either of two routes. By one route, the insulin A-chain and insulin B-chain are separately expressed and isolated, and the chains then are chemically combined to insulin. By the other route, a straight chain proinsulin precursor is expressed and isolated, and the product then is oxidatively renatured to proinsulin and the proinsulin enzymatically transformed to insulin.
Both of the above approaches to recombinant insulin production involve a similar sequence leading by chemical conversion and purification either to insulin A-chain S-sulfonate and insulin B-chain S-sulfonate ready for combination to insulin or to proinsulin S-sulfonate ready for disulfide interchange to proinsulin.
Any of the three S-sulfonates, insulin A-chain, insulin B-chain, or proinsulin, are in general obtained by the following sequence:
(1) Expression of product containing the desired peptide sequence joined at its amino terminal through a methionyl residue to an extraneous peptide sequence;
(2) Cleavage of the desired sequence from the extraneous portion using cyanogen bromide; and
(3) Sulfitolysis of the peptide cysteinyl thiols to produce the corresponding S-sulfonates.
It is essential, in making processes of this nature commercially feasible, to discover methods that will permit removal of sludge, salt, organic solvents, and other contaminants from the desired product (whether such product is the final product or an intermediate along the way) with little or no loss of such product.
A highly advantageous process which forms the basis of this invention has been discovered for enhancing the purity of proinsulin-like material from highly impure stocks thereof obtained via recombinant DNA methods. The process involves subjecting the impure stock to reverse phase purification on a macroporous acrylate ester copolymer resin support.